Tuberculosis (TB) is the leading cause of death among microbial agents in the world killing two million people annually. One-third of the world population is infected with Mycobacterium tuberculosis, the causative agent of the disease tuberculosis (TB). Immunocompromised individuals such as those diagnosed with HIV infections and the homeless who do not have access to medical care are at a greater risk for developing TB. In addiiton, although drug therapy for TB is available, compliance rates are low and have resulted in the emergence of multi-drug resistant strains. For these reasons, TB is again a public health threat and there is a renewal of scientific interest in the identification of virulence factors that aid in the pathogenesis of this organism. Mycobacterium tuberculosis is a facultative intracellular organism that resides and replicates within the macrophages of the host immune system. One of the ways the macrophage kills microorganisms is by releasing reactive oxygen and nitrogen species that are harmful to the microorganism. In this research proposal, genes coding for such enzymes as peroxiredoxins, glutaredoxin like "mycoredoxins", and nitrosothiol reductase that protect against oxidative and nitrosative stress and therefore aid in the survival of mycobacteria in macrophages will be investigated. In particular, enzymes requiring mycothiol, a unique thiol that is present exclusively in Actinomycetes and is essential to the mycobacterial cell, will be studied. M bovis BCG targeted mutants will be created in genes that encode these mycothiol dependent genes to validate gene function and to determine the role of these genes in pathogenesis. A transposon mutant library of M. bovis BCG will also be created and screened for mutants sensitive to disulfide stress which would result in the depletion of thiols in the cell. Mycobacterium bovis BCG, the vaccine strain for M. tuberculosis, is a model organism for studying M. tuberculosis virulence factors in Biosafety Level 2 containment.